In genetic screening studies of drosophila, ROBO1 has been identified as a molecule regulating the midline crossing of axons, and has been reported to work as a receptor for the Slit protein in subsequent studies (Kidd et al., Cell, 92, 205-215, 1998, Wang et al., Cell, 96, 771-784, 1999, Kidd et al., Cell, 96, 785-794, 1999, Brose et al., 96, 795-806, 1999). In addition, regarding the relationship between ROBO1 and cancer, the chromosome region 3p12 where the human ROBO1 gene is present is highly defective in lung cancer, and expression is suppressed by methylation of the ROBO1 promoter region in breast cancer and kidney cancer, suggesting that ROBO1 gene can be a tumor suppressor gene (Dallol et al., Oncogene, 21, 3020-3028, 2000). Indeed, hyperplasia of tracheal epithelial cells was observed in mice by deleting the first immunoglobulin region present at the N-terminus of ROBO1, which is similar to the minimal defect in the ROBO1 gene identified in lung cancer patients (Xian J et al., PNAS, 98, 15062-15066, 2001). Conversely, it has been reported that ROBO1 was expressed in new blood vessels of cancers, and that increased expression of Slit2, a ligand for ROBO1, on the cancer cells induces cancer neovascularization to direct cancer growth (Wang et al., Cancer Cell, 4, 19-29, 2003).
Meanwhile, the expression of the Slit2 gene, which is a ligand for ROBO1, is also suppressed in a number of cancer types by methylation or the like, and overexpression of Slit2 or addition of Slit2 induces a growth inhibition and apoptosis in lung cancer, breast cancer and large intestine cancer cells. These observation suggests that Slit2, a ligand for ROBO1, is also thought to be a tumor suppressor gene (Dallol et al., Cancer Research, 62, 5874-5880, 2002, Dallol et al., Cancer Research, 63, 1054-1058, 2003). However, it is not clear in this report through which receptor Slit2 exerts its effect in inhibiting the cell growth, thus the relationship between ROBO1 and cancer has not been absolutely clear.
Cancer is the most common cause of death in Japan. Among them, primary hepatocellular cancer is one type of cancer with poor prognosis, representing the third leading case of death (13%) in male and the fourth (9.0%) in female in 2001 (excerpt from “Population Dynamics Statistics”, Statistics and Information Department, Minster's Secretariat, Ministry of Health, Labour and Welfare). The number of chronic patients caused by viral infection is on the rise year after year and many of them lead to hepatic cirrhosis and then to hepatocellular cancers. Extremely strong demands exist for a diagnostic procedure at an early stage in the progression from hepatic cirrhosis to hepatocellular cancer and for a treatment of hepatocellular cancer. It is believed that without a groundbreaking solution, the number of deaths will follow an increasing trend in the 10 to 15 years to come.
Current hepatocellular cancer diagnostic procedure comprises a comprehensive evaluation based on biochemical data such as the serum value of GOP/GTP, alkaline phosphatase, albumin and the like, or a tumor marker AFP (a-fetoprotein), and diagnostic imaging. Then, if necessary, a small amount of tissue fragment is taken by needle biopsy for pathological judgment to confirm the diagnosis. Currently, tumor markers are mainly used for the diagnosis of hepatocellular cancer. The positive rate of alpha fetoprotein (AFP), which is the most common marker, is 60 to 70 percent in hepatocellular cancer patients, although it is sometimes also positive in chronic liver disease patients or pregnant female. Another hepatic cancer tumor marker PIVKA-II is positive in less than 50 percent of the patient, and the specificity for hepatocellular cancer is thought to be higher than AFP. Mainly these two examinations are currently in practice. In either case, false positive or double negative cases exist, thus a tumor marker with high specificity is needed.
Histological examination of the sample collected by needle biopsy is an important test for confirmed diagnosis of liver diseases. In particular, as the quantity of specimen may be limited, a more definite diagnosis technique is required. It is desired in the art to develop an antibody against an antigen specifically expressed in hepatic cancer to allow for not only pathological characteristics but also identification of hepatocellular cancer from a non-cancer tissue at an early stage.
In the current situation of diagnosis and monitoring of liver disease, progression from inflammation to fibrosis and malignant transformation are diagnosed by examination with multiple markers and examination by biopsy. In many hepatic cancer patients, the progression occurs from viral infection to hepatitis, chronic hepatitis, hepatic cirrhosis and then hepatic cancer. Consequently, a simple method for diagnosis and monitoring of liver diseases will be useful, not only in terms of healthcare economy, but also in mitigating the burden on the patients and in obtaining accurate medical guidelines.
Regarding treatment of hepatocellular cancer, many medical facilities are centered mainly on three types of therapy: surgical removal, transcatheter arterial embolization therapy, and percutaneous ethanol injection therapy. Either method has advantages and disadvantages, and even when transcatheter arterial embolization therapy is selected, which has a relatively broad application range and survival advantages, the rate of complete cure is currently thought to be on the order of 10%. Thus there is a great demand for a novel therapy.
Targeted therapy by monoclonal antibody against cancer specific tumor antigen provides a better outcome in breast cancer and in lymphoma and the like through an action mechanism different from conventional chemotherapy, although no clinical application has done for hepatocellular cancers yet. The action mechanisms of these antibody drugs include antibody dependent cytotoxicity (ADCC) via effector cells and complement-dependent cytotoxicity (CDC) via the complement, agonistic action by the function of the antibody itself, and the neutralization capability of the antibody. Recently molecular therapies have been applied in clinical sites. An antibody drug therapy which applies these molecular therapies and targets to a neoplasm-specifically expressed molecule found on hepatic cancer cells is expected to be developed in the future.
The following are documents related to the present invention: WO99/20764; WO98/48051; WO01/46697; WO03/29488; WO01/00828; WO01/57207; WO01/92581; WO02/04514; WO02/14500; WO02/29103.